Compositions of Less Immunogenic and Long-Circulating Protein-Lipid Complexes

ABSTRACT

Provided are lipidic particles comprising phosphatidylcholine, phosphatidylinositol and cholesterol. Also provided are compositions comprising the lipidic particles and having associated therewith therapeutic agents such as peptides, polypeptides or proteins. In these compositions, the therapeutic agents have reduced immunogenicity and/or longer circulating time. These compositions can be used for therapeutic administration of the peptides, polypeptides and/or proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/731,647, filed Mar. 30, 2007, now allowed, which in turn claimspriority to U.S. provisional patent application No. 60/870,177, filedDec. 15, 2006, and U.S. provisional patent application No. 60/787,411,filed Mar. 30, 2006, and U.S. provisional patent application No.60/787,586, filed Mar. 30, 2006, the disclosures of which areincorporated herein by reference.

This application is also a continuation of U.S. patent application Ser.No. 11/731,648, filed Mar. 30, 2007, now allowed, which in turn claimspriority to U.S. provisional patent application No. 60/870,177, filedDec. 15, 2006, and U.S. provisional patent application No. 60/787,411,filed Mar. 30, 2006, and U.S. provisional patent application No.60/787,586, filed Mar. 30, 2006, and U.S. provisional patent applicationNo. 60/865,062, filed Nov. 9, 2006, the disclosures of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by funding from the National Heart Lung andBlood Institute/National Institutes of Health Grant No. HL-70227. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In the treatment of diseased conditions, therapeutic interventions areoften undertaken which involve administration of foreign moleculeshaving therapeutically beneficial effects. However, such administrationscan often result in unwanted side effects resulting from activation ofthe body's immune response. Formation of antibodies followingadministration of therapeutics poses a serious clinical challenge. Theantibodies can abrogate activity and/or alter pharmaco-kinetics of thetherapeutic molecules.

This is particularly relevant when administering strong antigenicmolecules such as peptides, polypeptides or proteins. Many suchpolypeptides are routinely used as therapeutic molecules. For example,Factor VIII (FVIII) is an essential cofactor in the intrinsiccoagulation pathway. Any deficiency or dysfunction of FVIII results in ableeding disorder, characterized as hemophilia A. Replacement therapywith recombinant FVIII (rFVIII) or plasma-derived FVIII (pdFVIII) is thecommon therapy for controlling bleeding episodes. FVIII is a multidomainglycoprotein comprising of six domains (A1-A2-B-A3-C1-C2). Prior tosecretion into plasma, FVIII is subjected to proteolytic cleavage,leading to the generation of a heterodimer with molecular weightsranging from ˜170 to ˜300 KDa. The presence of the multiple proteolyticsites at the B domain level is responsible for the high heterogeneity ofthe FVIII preparations. In spite of being FVIII's largest domain (908amino acids residues or ˜40% of the total number of amino acidsresidues), the B domain lacks any essential function for the cofactorcoagulation activity. Deletion of the B domain leads to a lessheterogenic, genetically engineered rFVIII that corresponds to theshortest form of pdFVIII (e.g. ˜170 KDa). B domain deleted rFVIII(BDDrFVIII) is characterized by a higher specific activity than rFVIIIand can also be used for treatment of hemophilia.

Another therapeutic molecule is Factor VIIa (FVIIa). This is atrypsin-like serine protease which plays an important role in activatingthe extrinsic coagulation cascade. FVIIa is a poorly catalytic form offactor VII after the activating cleavage between Arg152 and Ile153. Uponinjury, circulating FVIIa becomes an efficient catalyst when forming acomplex with tissue factor (TF), its allosteric regulator that is foundon the outside of blood vessel. FVIIa-TF complex induces generation ofsmall amounts of thrombin which further triggers blood clotting. FactorVIIa has been approved by the Food and Drug Administration in the UnitedStates for uncontrollable bleeding in hemophilia A and B patients whohave developed inhibitory antibodies against replacement coagulationfactors, factor VIII and factor IX. Intravenous administration ofrecombinant human Factor FVIIa (rHu-FVIIa) has been introduced becauseof fewer side effects than other alternative treatment strategies and tocircumvent difficulty in preparing plasma-derived FVIIa. However, theshort circulation half-life of FVIIa requiring repeated bolus injectionsto achieve desired efficacy can be problematic.

Additionally, many other proteins are used as therapeutics. Theseinclude erythropoietin, VEG-F, other blood coagulation proteins,hormones (such as insulin and growth hormone) and the like. Strategiesthat can inhibit processing by immune system and also prolongcirculation time (reduce frequency of administration) would improveefficacy of proteins. Therefore there is a need in the area oftherapeutics to develop formulations that make the proteins lessimmunogenic, without significantly affecting the circulating time or theefficacy.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising therapeuticagents such that the immunogenicity of the agents is reduced and theircirculating time is increased. The compositions comprise lipidicparticles (also referred to herein as lipidic structures) comprisingphosphatidylcholine, phosphatidylinositol and cholesterol. Therapeuticagents such as peptides, polypeptides and/or proteins can be associatedwith the lipidic particles to form delivery compositions.

In these compositions, the therapeutic agent displays reducedimmunogenicity and longer circulating time.

In various embodiments, lipidic particles having associated therewithproteins such as Factor VIII, B domain deleted Factor VIII, Factor VII,lysozyme and Erythropoietin are disclosed.

In the description, the therapeutic agent associated with the lipidicparticles comprising PI is sometimes referred to as therapeuticagent-PI.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of biophysical and biochemicalcharacterization of Laurdan study of PC containing liposomes alone as aliquid or gel or PI containing lipidic particles with associated rFVIII.

FIG. 2. Biophysical and Biochemical characterization of rFVIII-PI (a):Transmission Electron Micrograph (TEM) of rFVIII-PI; (b); normalizedfluorescence emission spectra of free rFVIII and rFVIII-PI (1:10,000);(c): a list of capture monoclonal antibodies utilized in this study thattarget specific epitopes in rFVIII molecules; (d): the binding ofmonoclonal antibodies to rFVIII-PI at various lipid concentrations.Control is protein free liposomes. (e): far-UV CD spectra of rFVIII inthe presence (1:2,500) and in the absence of PI acquired at 20° C.; and(f): percent change in ellipticity of rFVIII as a function oftemperature in the presence and in the absence of PI.

FIG. 3. Effect of phosphatidylinositol on the Immunogenicity of rFVIII.(a, c) show the mean of total antibody titers (horizontal bars) andindividual (open circles) antibody titers were determined following s.c.and i.v. administrations, respectively. (b, d) show the mean ofinhibitory titers (horizontal bars) and individual (open circles)inhibitory titers were determined following s.c. and i.v.administrations, respectively.

FIG. 4. Influence of phosphatidylinositol on pharmacokinetics of rFVIII.The mean plasma concentration of rFVIII clotting activity after i.v.administration of free rFVIII and rFVIII-PI.

FIG. 5. Influence of phosphatidylinositol on pharmacokinetics ofBDDrFVIII. The mean plasma concentration of BDDrFVIII clotting activityafter i.v. administration of free BDDrFVIII and BDDrFVIII-PI.

FIG. 6. Acrylamide quenching for free lysozyme and lysozyme associatedwith PI containing lipidic particles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for lessimmunogenic and long circulating lipidic formulations for delivering atherapeutic agent. The formulations comprise a therapeutic agentassociated with lipidic structures comprising phosphatidyl choline (PC),and phosphatidyl inositol (PI) and cholesterol. The therapeutic agentmay be a peptide (generally 50 amino acids or less) a polypeptide(generally 100 amino acids or less) or proteins (larger than 100 aminoacids).

Although not intending to be bound by any particular theory, it isconsidered that the lower imunigenicity and/or the longer circulationtime is at least in part due to the lipidic particles having a uniquestructure. As seen under high magnification, the lipidic particles ofthe present invention do not appear to have donut like structurestypical of liposomal lamellarity. Substantial number of the lipidicparticles displayed disc like structures (see Example 2) which isattributable to reduced water volume thereby providing reduced contrastin the electron micrographs. Therefore the morphology appears differentfrom that of liposomes, possibly due to altered lipid structure andorganization, and reduced internal water volume. To further investigatethe lipid structure and organization we carried out fluorescence studiesusing Laurdan as probe. The probe partitions into the interfacial regionand the emission is sensitive to the presence and dynamics of watermolecules and lamellar structures of liposomes. The fluorescenceemission spectra were acquired for Laurdan labeled lipid particles ofthe present invention and also for liposomes, the latter serving ascontrol. For liposomes that undergo transition from gel to liquidcrystalline phase a red shift in the emission maxima, from 440 nm to 490nm is observed (FIG. 1). Based on the composition one would expect anemission spectrum corresponding to liquid crystalline phase. However,laurdan labeled lipidic particles of the present invention showed aspectrum that is neither gel like nor liquid crystalline like. Thus, thedata indicates that lamellar organization in this particle is differentfrom that of liposomes—possibly due to the water concentration anddynamics being altered in this particle. Centrifugation studies carriedout in discontinous dextran gradient indicated the particle floated morereadily than liposomes. Thus, the lipidic structures of the presentinvention appear to have altered lipid organization and dynamics,internal water volume, water concentration and/or dynamics near the headgroup compared to typical liposomes. In addition, the particle may belighter than the lamellar liposomes.

The association efficiency of the proteins in the lipidic particles aswell as the reduction in the immunogenicity of proteins associated withthe lipidic particles comprising PI was greater than for similarcompositions in which PI was replaced with PS, PA or PG. Since PI, PS,PA and PG are all anionic phospholipids, the advantage obtained by usingPI was surprising. Further because one of the proteins tested, FVIII isknown to bind more avidly to PS than to PI, it was surprising that theassociation efficiency of FVIII for PI containing lipidic structures washigher than that for PS containing liposomes.

The present invention also provides a method for preparing the lipidicstructures. The lipidic structures can be prepared by thin lipid filmhydration using the appropriate molar ratios of PC, PI and cholesterolin a suitable buffer. The lipids are dissolved in chloroform and thesolvent is dried. The resulting multilamellar vesicles (MLVs) areextruded through the desired size filters (sizing device) under highpressure to obtain lipidic structures of the present invention. It isgenerally preferred that the size of the lipidic particles should beless than 140 nm (as calculated from micrographs and dynamic lightscattering measurements) so that the particles are not filtered out inthe Reticulo Endothelial System (RES) so as to become available for theimmune system reaction. Thus it is preferred to have at least 50% of theparticles to be less than 140 nm More preferably, the particles shouldbe less than 120 nm and still more preferably between 40 and 100 nm. Invarious embodiments, 50, 60, 70, 80, ad 90% of the particles are lessthan 140 nm and more preferably between 40 and 100 nm.

To effect association of the protein with the lipidic structures, theprotein in a suitable buffer is added to the lipidic structures. Thefree protein is then separated from the lipidic structures by routinecentrifugation methods such as density gradient centrifugations. Invarious embodiments, the association efficiency of the protein with thelipidic particles is at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90and 95%. It desired, the lipidic particles with the associatedtherapeutic agent can be lyophilized for future use.

In one embodiment, the lipidic structures of the present invention priorto association with the protein can be lyophilized and stored. Whenneeded, the lipidic structures can be reconstituted and then used forcombination with protein to effect association of the protein with thelipidic structures prior to use.

The present invention can be used for association of therapeutic agentssuch as proteins, polypeptides or peptides with the lipidic structures.The protein and peptides with wide biochemical properties can be loadedin the particles. The proteins may be neutral or charged (negatively orpositively). Such proteins include proteins involved in the bloodcoagulation cascade including Factor VIII (FVIII), Factor VII (FVII),Factor IX (FIX), Factor V (FV), and von Willebrand Factor (vWF), vonHeldebrant Factor, tissue plasminogen activator, insulin, growthhormone, erythropoietin alpha, VEG-F, Thrombopoietin, lysozyme and thelike.

The ratio of PC to PI to cholesterol can be between 30:70:1 to 70:30:33.Thus the ratio of PC to PI can vary between 30:70 to 70:30. In oneembodiment, it is between 40:60: to 60:40 and in another embodiment45:55 to 55:45. In another embodiment, it is 50:50. The cholesterol (asa percentage of PC and PI together) is between 1 and 33% as structuresformed at higher cholesterol ratio than 33% lack stability. In oneembodiment, the cholesterol is 5-15%.

The association of the protein with the lipidc structures can be suchthat the molar ratio between the protein to lipid is between 1:200(protein:lipid) to 1:30,000 (protein:lipid). In one embodiment it isabout 1:10,000 (protein:lipid). In other embodiments, the ratio is about1:2,000 or 1:4,000.

The phospholipids PC and PI have two acyl chains. The length of the acylchains attached to the glycerol backbone varies in length from 12 to 22carbon atoms. The acyl chains may be saturated or unsaturated and may besame or different lengths. Some non-limiting examples of 12-22 carbonatom saturated and unsaturated acyl chains are shown in Tables 1A and1B:

TABLE 1A Symbol Common Name Systematic name Structure 12:0 Lauric aciddodecanoic acid CH₃(CH₂)₁₀COOH 14:0 Myristic acid tetradecanoic acidCH₃(CH₂)₁₂COOH 16:0 Palmitic acid hexadecanoic acid CH₃(CH₂)₁₄COOH 18:0Stearic acid octadecanoic acid CH₃(CH₂)₁₆COOH 20:0 Arachidic acideicosanoic acid CH₃(CH₂)₁₈COOH 22:0 Behenic acid docosanoic acidCH₃(CH₂)₂₀COOH

TABLE 1B Symbol Common Name Systematic name Structure 18:1 Oleic acid9-Octadecenoic CH₃(CH₂)₇CH═CH(CH₂)₇COOH acid 16:1 Palmitoleic acid9-Hexadecenoic CH₃(CH₂)₅CH═CH(CH₂)₇COOH acid 18:2 Linoleic acid 9,12-CH₃(CH₂)₄(CH═CHCH₂)₂(CH₂)₆COOH Octadecadienoic acid 20:4 Arachidonicacid 5,8,11,14- CH₃(CH₂)₄(CH═CHCH₂)₄(CH₂)₂COOH Eicosatetraenoic acid

The acyl chains attached to PC are preferably 12 to 22. These can besaturated or unsaturated and can be same or different length. The acylchains attached to PI can be from 12 to 22 and can be saturated orunsaturated. The chains of the PC and the PI can be same or aredifferent in length.

The PC and PI can be obtained from various sources both natural andsynthetic. For example, soy PI and egg PC are available commercially.Additionally, synthetic PC and PI are also available commercially.

The compositions can be delivered by any standard route such asintravenous, intramuscular, intraperitonial, mucosal, subcutaneous,transdermal, intradermal, oral or the like.

The invention is described by the following examples, which are intendedto be illustrative and not restrictive in any way.

Example 1

This example describes the preparation of the lipidic particles.

Materials: Albumin free full-length rFVIII (Baxter Health Care Glendale,Calif.) was used as antigen. Advate was provided as a gift from WesternNew York Hemophilia foundation. Dimyristoyl phosphatidylcholine (DMPC)and soybean phosphatidylinositol (Soy PI) were purchased from AvantiPolar Lipids (Alabaster, Ala.). Cholesterol, IgG-free bovine serumalbumin (BSA), and diethanolamine were purchased from Sigma (St. Louis,Mo.). Goat antimouse-Ig and antirat-Ig, alkaline phosphatase conjugateswere obtained from Southern Biotechnology Associates, Inc. (Birmingham,Ala.). p-Nitrophenyl phosphate disodium salt was purchased from Pierce(Rockford, Ill.). Monoclonal antibodies ESH4, ESH5, and ESH8 werepurchased from American Diagnostica Inc. (Greenwich, Conn.). Monoclonalantibody N77210M was purchased from Biodesign International (Saco, Me.).Normal coagulation control plasma and FVIII-deficient plasma werepurchased from Trinity Biotech (County Wicklow, Ireland). The CoamaticFactor VIII kit from DiaPharma Group (West Chester, Ohio) was used todetermine the rFVIII activity in plasma samples.

Preparation of rFVIII-PI lipidic particles: The particles were preparedby thin lipid film hydration of appropriate molar ratios of DMPC, soyPI, and cholesterol (50:50:5) with Tris buffer (TB) (25 mM Tris, and 300mM NaCl, pH=7.0). The required amount of lipids was dissolved inchloroform in a kimax tube and the solvent was dried using Buchi-R200rotaevaporator (Fisher Scientific, NJ). Multilamellar vesicles (MLV)were formed by vortex, mixing the lipid dispersions at 37° C. for 20min. The resulting MLV were extruded through double polycarbonatemembranes of 80 nm pore size (GE Osmonics Labstore, Minnetonka, Minn.)in a high-pressure extruder (Mico, Inc., Middleton, Wis.) at a pressureof ˜250 psi and then sterile-filtered through a 0.22 um Millex™-GPfilter unit (Millipore Corporation, Bedford, Mass.). Phosphate assay wasused to estimate phospholipids concentrations. Particle size wasmonitored using a Nicomp Model CW 380 particle size analyzer (ParticleSizing Systems, Santa Barbara, Calif.). The protein was added to thelipid particles at 37° C. and during this process Ca²⁺ ion concentrationwas decreased from 5 mM CaCl₂ to 0.2 mM CaCl₂ in TB to ensure optimallipid-Ca²⁺ interaction and possible lipid phase change. The protein tolipid ratio was maintained at 1:10,000 for all experiments, unlessstated otherwise.

Separation of Free rFVIII from rFVIII-PI: Discontinuous dextran densitygradient centrifugation technique was used to separate the free proteinfrom the lipidic particles. Briefly, 0.5 ml of incubated rFVIII-PImixture was mixed with 1.0 ml of 20% (w/v) dextran (in Ca⁺² free TB) ina 5 ml polypropylene centrifuge tube. 3 ml of 10% (w/v) dextran was thencarefully added on top of the mixture followed by 0.5 ml of Ca⁺² freeTB. After centrifugation at 45,000 rpm at 4° C. for 30 min in a BeckmanSW50.1 rotor, the bound protein and free lipidic particles would floatto the top of the dextran band unbound rFVIII would stay at the bottomof the gradient. One-stage activated partial thromboplastin time (APTT)assay was used to estimate the protein association efficiency ofrFVIII-PI. This procedure yielded an association efficiency of 72±9% andis much higher than that observed with Phosphatidyl Serine (PS)containing liposomes (45±16.8%) (Purohit, et al. Biochim Biophys Acta1617, 31-38 (2003). Kemball-Cook, et al., Thromb Res 67, 57-71 (1992)).

Example 2

This example describes characterization of the lipid structures preparedin Example 1. The lipid dispersion samples for microscopic analysis wereprepared by air-drying on formvar-coated grids and negatively stainingthem with 2% uranyl acetate for approximately 1 min. The samples werephotographed using a Hitachi H500 TEM operating at 75 kV. Negatives werescanned at 300 dpi with an Agfa Duoscan T1200 scanner. The morphology ofthe particles determined using Transmission electron microscopic studiesindicated the following. The particle size was found to be around 100nm, consistent with dynamic light scattering studies (data not shown).The analysis of the micrograph showed that the donut like structurestypical of liposomal lamellarity was not observed instead particlesdisplayed disc like structures (FIG. 2 a) and it is possible that uniquelipid organization distinct from liposomes are formed that canaccommodate higher mol % of FVIII.

Example 3

This example describes fluorescence analysis of rFVIII and rFVIII-PI.The effect of PI on the tertiary structure of rFVIII was determined byexciting the samples either at 280 nm or at 265 nm and the emission wasmonitored in the wavelength range of 300-400 nm. The spectra wereacquired on a PTI-Quantamaster fluorescence spectrophotometer (PhotonTechnology International, Lawrenceville, N.J.). Protein concentrationwas 5 ug/ml and slit width was set at 4 nm. The fluorescence emissionspectra of FVIII loaded in this particle showed blue shifted emissionmaxima compared to free protein suggesting that FVIII is located inhydrophobic environment (FIG. 2 b). This observation is in contrast tofluorescence spectrum observed for FVIII associated with PS containingliposomes where no change in fluorescence properties was observed(Purohit et al., 2003) and is consistent with molecular model proposedbased on crystallographic and biophysical studies. FVIII was associatedwith PS containing liposomes only via the C-terminal region (2303-2332)of C2 domain and the rest of the molecule is accessible to bulk water(Purohit et al., 2003). However, in FVIII-PI particulates, it is likelythat most of the molecular surface of FVIII is buried in hydrophobicacyl chain region of the lipidic particle and/or the protein is locatedat the lipid-water interface where water concentration at lipidinterface may be less for PI particles.

Example 4

This example describes Sandwich ELISA and detection of rFVIII epitopesinvolved in rFVIII-PI association. In order to determine the rFVIIIepitopes that were associated with PI, sandwich ELISAs were performed.Briefly, Nunc-Maxisorb 96-well plates were coated overnight at 4° C.with appropriate concentrations of capture monoclonal antibodies incarbonate buffer (0.2 M, pH 9.6). Plates were then washed with Tween-PBS(2.7 mM KCl, 140 mM NaCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄.2H₂O, 0.05% w/vTween 20, pH 7.4) and then blocked with 1% BSA (prepared in PBS) for 2 hat room temperature. 100 ul of 0.5 ug/ml of various dilutions ofrFVIII-PI (1:0,1:5,000, 10,000, and 50,000) or PI-containing liposomesin blocking buffer were incubated at 37° C. for 1 h. Plates were washedand then incubated with 100 ul of a 1:500 dilution of rat polyclonalantibody containing a 1:1,000 dilution of goat antirat-Ig-alkalinephosphatase conjugate in blocking buffer at room temperature for 1 h.After the last wash, 200 ul of a 1 mg/ml p-nitrophenyl phosphatesolution in diethanolamine buffer (1 M diethanolamine, 0.5 mM MgCl₂) wasadded and incubated for 30 min at room temperature. 100 ul of 3 N NaOHwere added to stop the reaction. A plate reader was used to measure theoptical density at 405 nm.

In order to investigate the molecular surface area associated with PI,sandwich ELISA studies were carried out (FIGS. 2 c and 2 d). Therationale for this experiment is that domains associated with lipidicparticle are shielded and hence will not be available for monoclonalantibody binding. Therefore, sandwich ELISA is an indirect, qualitativemethod to provide insight into protein surface accessible to bulkaqueous compartment. The binding of FVIII in the absence of PI wasnormalized to 100% to account for differences in binding affinity ofvarious antibodies and decrease in antibody binding in the presence ofPI was interpreted as domains of FVIII involved in PI binding.PhosphatidylCholine (PC) vesicles were used as negative control as theassociation efficiency of FVIII in PC vesicles is around 10±4% (Purohitet al., 2003. Biochim Biophys Acta, 1617:31-38). PS liposomes were usedas positive control for binding of C2 domain antibodies based oncrystallographic and biophysical/biochemical studies. It has been shownthat C-terminal region of the C2 domain involving 2303-2332 is involvedin lipid binding and the A2 domain is further apart from the liposomesurface (Stoilova-McPhie et al., 2002, Blood, 99:1215-1223). Based onthis molecular topology, C2 and A2 domains are spatially well separatedand only lipid-binding region in C2 domain may be shielded from antibodybinding due to liposome association (Purohit et al. 2003;Stoilova-McPhie et al., 2002). Monoclonal antibodies directed against C2and A2 domains were chosen based on this molecular model of FVIII boundto PS containing liposomes. The results indicated that the molecularsurface of FVIII that is in contact with PI is different from thatobserved for PS. In PC liposomes no lipid concentration dependent changein antibody binding was observed indicating that no specific bindingbetween FVIII and PC, whereas for PS vesicles, lipid concentrationdependent changes observed only for antibody directed against the lipidbinding domain (ESH 4), consistent with the model proposed based oncrystallographic studies. However, for FVIII-PI, all the monoclonalantibodies used in this study showed reduced binding and was dependenton PI concentration (FIG. 2 d). The results indicated that both C2 andA2 domains are somewhat inaccessible for antibody binding possibly dueto steric hindrance and/or substantial surface area of the FVIIImolecule is buried in the PI particle.

Example 5

This example describes CD analysis of rFVIII and rFVIII-PI. CD spectrawere acquired on a JASCO-715 spectropolarimeter calibrated with d-10camphor sulfonic acid. The protein to lipid ratio was 1 to 2,500 wherethe protein concentration used was 20 ug/ml (98.6 IU/ml). Spectra wereobtained over the range of 255 to 208 nm for secondary structuralanalysis using a 10 mm quartz cuvette. Thermal denaturation of therFVIII and rFVIII-PI was determined by monitoring the ellipticity at 215nm from 20 to 80° C. using a heating rate of 60° C./h. The lightscattering effect due to the presence of lipidic particles was correctedas described previously (Balasubramanian et al., 2000, Pharm Res 17,344-350).

The CD studies showed that such molecular topology did not alter thesecondary structure of the protein, as the CD spectrum of the proteinwas not changed by PI association (FIG. 2 e). Thermal unfolding is oftenused to investigate the intrinsic stability. FVIII associated with PInano particle displayed a shallower melting with Tm slightly higher thanthat observed for FVIII indicating that the association of FVIII with PIimproved the intrinsic stability of FVIII (FIG. 2 f).

Example 6

The association of the protein was carried out in several buffer systemsas shown in Tables 2A-2C. In all the cases the protein to lipid ratiowas 1:10,000. The free protein was then separated from the lipidicstructures by density gradient centrifugations and the proteinassociated with each fraction was measured using activity andspectroscopic assay.

Table 2A shows the percentage of rFVIII associated with 50% DMPC:50%SPI:5% Chol (100 nm) with different buffer compositions at 37° C. Table2B shows the percentage of rFVIII associated with 50% DMPC:50% SPI (100nm) with different buffer compositions at 37° C. Table 2C: Percentage ofrFVIII associated with 50% DMPC:50% SPI:15% Chol (100 nm) with differentbuffer compositions at 37° C.

TABLE 2A Percent Buffer Composition Association 300 mM NaCl, 25 mM Tris,pH 7.4 70~80 PBS, pH 7.0 95 50 mM Tris, 150 mM NaCl, 3 mM NaN₃, pH 7.496 25 mM Tris, pH 7.4 42 10 mM Hepes, pH 7.4 23

TABLE 2B Percent Buffer Composition Association 300 mM NaCl, 25 mM Tris,pH 7.4 67 PBS, pH 7.0 88 50 mM Tris, 150 mM NaCl, 3 mM NaN₃, pH 7.4 5810 mM Hepes, pH 7.4 33

TABLE 2C Percent Buffer Composition Association 300 mM NaCl, 25 mM Tris,pH 7.4 66 PBS, pH 7.0 93 50 mM Tris, 150 mM NaCl, 3 mM NaN₃, pH 7.4 5110 mM Hepes, pH 7.4 32

Example 7

This example describes immunogenicity studies: Breeding pairs ofhemophilia A mice (C57BL/6J) with a target deletion in exon 16 of theFVIII gene were used. A colony of hemophilia A mice was established andanimals aged from 8-12 weeks were used for the in vivo studies. Sincethe sex of the mice has no impact on the immune response, both male andfemale mice were used for the studies.

The relative immunogenicity of free rFVIII and rFVIII-PI were determinedin hemophilia A mice. This mouse model is suitable for investigatingimmunogenicity of FVIII as the antibody response patterns against FVIIIare very similar to those observed in hemophilic patients. 8 male and 10female mice received 4 weekly intravenous injections (via penile vein)and subcutaneous injections of 10 IU of FVIII (400 IU/kg), respectively.2 weeks after the last injection, blood samples were collected in acidcitrate dextrose (ACD) buffer (85 mM sodium citrate, 110 mM D-glucoseand 71 mM citric acid) at a 10:1 (v/v) ratio by cardiac puncture. Plasmawas separated by centrifugation at 5,000 rpm at 4° C. for 5 min. Sampleswere stored at −80° C. immediately after centrifugation. The totaltiters were determined by ELISA studies and the inhibitory titer wasdetermined using a modified Bethesda assay as described previously(verbruggen et al., Thromb Haemost, 1995, 73:2470251).

The results showed that PI reduced antibody response in Hemophilia mice(FIG. 3). Animals treated with rFVIII-PI displayed significantly lowertotal antibody titers (FIGS. 3 a and 3 c) compared to animals treatedwith rFVIII alone. Titers were 2379±556 (±S.E.M; n=10) for FVIII-PIgiven by sc route, compared to 13,167±2042 (n=15) for animals treatedwith rFVIII alone. These differences were significant at P<0.05. Animalstreated with rFVIII-PI by i.v. also showed lower mean antibody titers;for FVIII-PI antibody titers were found to be of 3321±874 (n=8) andthose treated with rFVIII had titers of 4569±1021 (n=8) and thisdifference was not significant. However, the inhibitory titers thatabrogate the activity of the protein reduced significantly for FVIII-PIgiven by both sc and iv route (FIGS. 3 b and 3 d). For scadministration, the inhibitory titers reduced by more than 70%. Foranimals given FVIII alone by iv route, the inhibitory titers were 675±71and it reduced to 385±84 for FVIII-PI and this reduction isstatistically significant at p<0.05. Together these results indicatethat PI containing lipidic particles not only reduced overallanti-rFVIII antibody titers, but also lowered the titer of antibodiesthat abrogate the activity of the protein.

Example 8

This example describes Pharmacokinetics Studies. rFVIII or rFVIII-PI (10IU/25 g) was administered to male hemophilia A mice as a singleintravenous bolus injection via penile vein. Blood samples werecollected in syringes containing ACD buffer (10:1 v/v) at 0.08, 0.5, 1,2, 4, 8, 16, 24, 36, and 48 h after the injections by cardiac puncture(n=3 mice/time point). Plasma was collected immediately bycentrifugation (5,000 rpm, 5 min, 4° C.) and stored at −70° C. untilanalysis. Chromogenic assay was used to measure the activity of rFVIIIin plasma samples. The average values of rFVIII activities at each timepoint were used to compute basic pharmacokinetic parameters (half-life,MRT and area under the plasma activity curve) using a noncompartmentalanalysis (NCA)²⁰ (WinNonlin Pharsight Corporation, Mountainview,Calif.). The areas under the plasma activity (AUC) versus time curvesfrom 0 to the last measurable activity time point were measured bylog-linear trapezoidal method. The elimination rate constant (lambda z)was estimated by log-linear regression of the terminal phaseconcentration. The elimination half-life (t_(1/2)) was calculated as ln2/lambda z and MRT was calculated from AUMC/AUC where AUMC is the areaunder the curve plot of the product of concentration and time versustime.

Data was analyzed by ANOVA using Minitab (Minitab Inc., State College,Pa.). Statistical difference (p<0.05) was detected by the Studentindependent t-test, and one-way ANOVA followed by Dunnette's post-hocmultiple comparison test. For PK studies, repeated-measures ANOVA wasused to compare the profiles generated by the two treatments.Bailer-Satterthwaite method was used to compare differences in systemicexposure between the two treatments.

The MRT and AUC is found to be higher for FVIII-PI compared to FVIII andalso showed prolonged terminal elimination phase. The circulationhalf-life of FVIII associated with PI lipidic particle (7.6 hrs) ishigher than that observed for free FVIII (2.3 hrs). Substantial proteinactivity was detected after 48 hrs of injection for animals that aregiven FVIII-PI particles; in contrast no detectable FVIII activity wasobserved at 48 hrs in animals that received FVIII alone (FIG. 4).Further, the protein activity was detectable for only 24 hrs followingadministration of the PS containing liposomes (data not shown) and isdue to the rapid uptake of PS liposomes by the RES. However, in thepresence of PI it is possible that the cellular uptake is reduced and isconsistent with the stealth like properties of PI.

Example 9

This examples describes the Association efficiency for rFVIII and atruncated version of FVIII. BDDrFVIII. The lipidic structures havingFVIII or BDDrFVIII were prepared by a process as described above. Thepercent association for these proteins is shown below in Table 3.

TABLE 3 Percentage of rFVIII associated with lipidic particlesof variouscompositions. Lipidic particle Temper- % Protein Composition Size atureAssoci- (protein:lipid) (molar ratio) (nm) (° C.) ation rFVIII DMPC:SPI200 37 30.7 (1:10,000) (70:30) rFVIII DMPC:SPI 100 37 43.9 (1:10,000)(70:30) rFVIII DMPC:SPI 100 37 74.6 (1:10,000) (50:50) rFVIIIDMPC:SPI:Chol 100 37 30.4 (1:10,000) (70:30:5) rFVIII DMPC:SPI:Chol 10037 81.2 (1:10,000) (50:50:5) rFVIII DMPC:SPI:Chol 100 20 61.8 (1:10,000)(50:50:5) BDDrFVIII DMPC:SPI:Chol 100 37 59.0 (1:10,000) (50:50:5)BDDrFVIII DMPC:SPI:Chol 100 20 44.7 (1:10,000) (50:50:5) BDDrFVIIIDMPC:SPI:Chol 100 37 65.0 (1:20,000) (50:50:5) BDDrFVIII DMPC:SPI:Chol100 20 61.3 (1:20,000) (50:50:5)

As seen above, DMPC:SPI:Cholesterol (50:50:5) formulation showed highestassociation efficiency than other formulations for both rFVIII andBDDrFVIII. Cholesterol is preferably included in the formulation toincrease liposome stability in plasma. Size of liposome and associationtemperature, and lipid concentration all play important roles inassociation. DMPC:SPI:Cholesterol (50:50:5) formulation surprisingly hashigher association for rFVIII than BDDrFVIII even though BDDrFVIII haslower Molecular weight and size. Conformational changes as a result ofthe B domain deletion could be responsible for a decrease in the bindingaffinity of BDDrFVIII towards PI containing lipidic particles.

Pharmacokinetics Studies

Male hemophilic mice (20-24 g, 22-25 weeks old) were given 10 IU/25 g ofrBDDFVIII associated with the lipidic structures (referred to asPI-BDDrFVIII) as a single i.v. bolus injection via the penile vein.Blood samples were collected 0.08, 0.5, 1, 2, 4, 8, 16, 24, 36, and 48hr post dose by cardiac puncture (n=1/time point) and added to acidcitrate dextrose (ACD) at a 10:1 (v/v) ratio. Plasma was separated bycentrifugation at 5000 g for 5 min at 4° C. and stored at −70° C. untilanalysis. Plasma samples were analyzed for the activity of the proteinby the chromogenic assay. The activities of BDDrFVIII determined at eachtime point were then utilized to estimate basic PK parameters(half-life, t1/2 and area under the plasma activity curve/exposure,AUAC)

The PK Normalized PK Profile for Free BDDrFVIII and PI-BDDrFVIIIFormulation (n=1) is shown in FIG. 5. The Area Under the Curve AUC(hr*IU/mL) or Free BDDrVIII was 955.1 and for BDDFVIII associated withlipidic structures was 1058.7.

Example 10

This example describes the preparation of PS containing liposomes thatwere used for comparison with the PI containing lipidic structures inother Examples. BDDrFVIII was mixed with solution containing differentconcentration of O-phospho-L-serine (OPLS) or phosphocholine in such away that the final protein concentration is maintained constant at 3ug/mL. The OPLS or phosphocholine concentration vared between 0 and 100uM. Each mixture is incubated for 5 minutes before subjecting to furtheranalysis.

The intrinsic fluorescent of BDDrFVIII in the presence of increasingconcentration of phospholipids head group was measured with a QuantaMaster PTI instrument. The excitation was set to 285 nm and the emissionwas recorded at peak maximum (e.g. 330 nm). The normalized fluorescence(F/F₀) data was plotted vs. [lipid] and used for the determination ofthe dissociation constant for lipid head group-BDDrFVIII interaction.

To monitor the aggregation process of BDDrFVIII in the presence andabsence of OPLS and PC headgroup, the sample fluorescence anisotropy wasmeasured as a function of temperature using the a Quanta Master PTIspectrofluorometer equipped with motorized polarizer prisms. The datawas plotted as anisotropy vs. temperature and fitted to a sigmoidalcurve. The inflection point of each curve was obtained.

The dissociation constant (Kd (uM)) for OPLS was 70.2 and that forphosphocholine was 24.2. The inflection point for free BDDrFVIII was71.6, for BDDrFVIII in OPLS liposomes was 79.4 and for BDDrFVIII inphosphocholine liposomes was 72.2.

To study the immunological properties of BDDrFVIII-OPLS complex, 8 to 12week old hemophilic mice received 4 weekly injection containingdifferent BDDrFVIII formulations (free BDDrFVIII anf BDDrFVIII-OPLScomplex—prepared as described above; [OPLS]=10 mM). The dose for eachinjection is of 10 IU/animal. Two weeks following the last injection,blood samples were collected and analyzed for the presence of inhibitoryantibodies using a modified Bethesda assay. Results indicated astatistical significant decrease in the immune response for theBDDrFVIII-OPLS complex compared to free BDDrFVIII (P<0.05).

The association efficiency for rFVIII andBDDrFVIII-phosphatidylserine-containing liposomes was determined.Dimyristoylphosphatidylcholine (DMPC):brain phosphatidylserine (BPS)liposomes (molar ratio 70:30). The required amounts of DMPC and BPS weredissolved in chloroform. A thin lipid film was formed on the walls of aglass tube, by removing the solvent in a Buchi-R200 rotoevaporator(Fisher Scientific). The liposomes were prepared by rehydration of thelipid film with Tris buffer (TB 25 mm Tris, 300 mM NaCl, 5 mM CaCl₂pH=7.4) at 37° C. The liposomes were extruded eight times through doublestacked 100 nm or 200 nm polycarbonate membranes using a high pressureextruder (Lipex Biomembranes, Inc.) at a pressure of ˜200 psi. The sizedistribution of the liposomes was monitored using a Nicomp model CW380size analyzer (Particle Sizing System).

Liposomal Protein Preparation

The association of the protein with the preformed liposomes was achievedby incubating the protein in the presence of the liposomes at 37° C. for30 minutes with occasional gentle swirling. The protein to molar ratiowas maintained the same for all preparation (1:10,000).

PEGylation of Preformed Protein-Liposome Mixtures.

PEGylation of the preformed liposomes was achieved by addition of theliposomal preparations to a dry powder of 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DMPC-PEG 2000) or 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DSPE-PEG 2000). The incubation was performed for 45 minutes atroom temperature. Care was taken to maintain the DMPC-PEG 2000concentration below the critical micellar concentration in order tofacilitate the transfer of DMPC-PEG 2000 to the preformed lipidicbilayer.

Separation of Free Protein from Liposome Associated Protein

To estimate the amount of protein associated with liposomes, freeprotein was separated from liposome-associated protein by floatation ona discontinuous dextran density gradient. Briefly, 0.5 ml of theliposome-protein mixture was mixed with 1 ml of 20% (w/v) dextran (incalcium free Tris buffer) in a 5 ml polypropylene centrifuge tube and 3ml of 10% (w/v) dextran and 0.5 ml calcium free Tris buffer wereoverlaid on the liposome-containing band. The gradient was subjected toultracentrifugation at 45,000 rpm for 30 min in a Beckman SW50.1 rotor.The liposomes and their associated protein floated to the interface ofthe buffer/10% dextran bands, and the unassociated protein remained atthe bottom. The activity of the protein associated with liposomes wasdetermined using the one-stage activated partial thromboplastin time(APTT) assay. Results are shown in Table 4A and 4B.

TABLE 4A Association efficiency % Non-PEGylated liposomes BDDrFVIII 200nm DMPC:BPS (70:30) 41.5 BDDrFVIII 100 nm DMPC:BPS (70:30) 44.4

TABLE 4B PEGylated liposomes rFVIII (Control) 200 nm DMPC:BPS (70:30)28.0 BDDrFVIII 200 nm DMPC:BPS (70:30) 38.0 BDDrFVIII 100 nm DMPC:BPS(70:30) 48.3

BDDrFVIII retains all critical structural characteristics of the parentmolecule, including the binding properties towards phosphatidylserine(PS) containing lipidic membranes as well as its activity. Theassociation efficiency of BDDrFVIII was higher than that observed forfull length rFVIII.

Immunological properties of BDDrFVIII-PS containing liposomes were alsotested. Eight to 12 week old hemophilic mice received 4 weeklyinjections containing 10 IU of BDDrFVIII-PS containing liposomes(prepared as described in example 3). Two weeks following the lastinjection, blood samples were collected and analyzed for the presence ofinhibitory antibodies using a modified Bethesda assay.

Example 11

This example describes the association efficiency for BDDrFVIIIassociated with PS and phosphatidylethanolamine (PE) containingliposomes which were used for comparison purposes.

Liposome Preparation.

DMPC:BPS:dioleoylphosphatidylethanolamine (DOPE) (molar ratio 70:10:20)were prepared as described bellow: The required amounts of DMPC, BPS andDOPE were dissolved in chloroform. A thin lipid film was formed on thewalls of a glass tube, by removing the solvent in a Buchi-R200rotoevaporator (Fisher Scientific). The liposomes were prepared byrehydration of the lipid film with Tris buffer (TB 25 mm Tris, 300 mMNaCl, 5 mM CaCl₂ pH=7.4) at 37° C. The liposomes were extruded eighttimes through double stacked 100 nm polycarbonate membranes using a highpressure extruder (Lipex Biomembranes, Inc.) at a pressure of ˜200 psi.The size distribution of the particles was monitored using a Nicompmodel CW380 size analyzer (Particle Sizing System).

Liposomal Protein Preparation

The association of the protein with the preformed liposomes was achievedby incubating the protein in the presence of the liposomes at 37° C. for30 minutes with occasional gentle swirling. The protein to molar ratiowas maintained the same for all preparation (1:10,000).

PEGylation of Preformed Protein-Liposome Mixtures.

PEGylation of the preformed liposomes was achieved by addition of theliposomal preparations to a dry powder of 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DMPC-PEG 2000) or 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DSPE-PEG 2000). The incubation was performed for 45 minutes atroom temperature. Care was taken to maintain the DMPC-PEG 2000concentration below the critical micellar concentration in order tofacilitate the transfer of DMPC-PEG 2000 to the preformed lipidicbilayer.

Separation of Free Protein from Liposome Associated Protein

To estimate the amount of protein associated with liposomes, freeprotein was separated from liposome-associated protein by floatation ona discontinuous dextran density gradient. Briefly, 0.5 ml of theliposome-protein mixture was mixed with 1 ml of 20% (w/v) dextran (incalcium free Tris buffer) in a 5 ml polypropylene centrifuge tube and 3ml of 10% (w/v) dextran and 0.5 ml calcium free Tris buffer wereoverlaid on the liposome-containing band. The gradient was subjected toultracentrifugation at 45,000 rpm for 30 min in a Beckman SW50.1 rotor.The liposomes and their associated protein floated to the interface ofthe buffer/10% dextran bands, and the unassociated protein remained atthe bottom. The activity of the protein associated with liposomes wasdetermined using the one-stage activated partial thromboplastin time(APTT) assay. Results are shown in Table 5.

TABLE 5 Exp Percent # size composition association I Control 100 nmDMPC:BPS:DOPE (90:10:00) 18.0 II 100 nm DMPC:BPS:DOPE (70:10:20) 57.4III 100 nm DMPC:BPS (70:30) 44.4 IV 100 nm DMPC:BPS:DOPE (70:10:20) +45.6 3% PEG

PS containing liposomes are rapidly cleared from circulation by thereticuloendothelium system (RES). Phosphatidylethanolamine (PE)increases the affinity of FVIII towards PS containing lipids. In thepresent example, PE is added to the composition at the expense of PS.The association efficiency is found to increase with increasingconcentration of PE (compare Exp #I and II). In the absence of PEG, theassociation efficiency is found to be higher for DOPE containingparticles than in the absence of DOPE (Exp #II and III). Decreasing thecontent of PS in the formulation while achieving a higher associationefficiency is more beneficial from the perspective of pharmacologicalproperties of BDDrFVIII.

Example 12

This example describes the application of this method to anotherprotein, Factor VII. In this example, lipidic structures comprising FVIIwere prepared. The required amounts of DMPC, SPI and Chol(Dimyristoylphosphatidylcholine (DMPC):soy phosphatidylinositol(SPI):Cholesterol (Chol) (molar ratio 50:50:5) were dissolved inchloroform. A thin lipid film was formed on the walls of a glass tube,by removing the solvent in a Buchi-R200 rotoevaporator (FisherScientific). The lipidic particles (LP) were prepared by rehydration ofthe lipid film with 25 mM tris buffer (300 mM NaCl, pH=7.0; calciumfree) at 37° C. The LP were extruded twenty times through double stacked80 nm polycarbonate membranes using a high pressure extruder (LipexBiomembranes, Inc.) at a pressure of ˜200 psi. The size distribution ofthe particles was monitored using a Nicomp model CW380 size analyzer(Particle Sizing System).

The association of the protein with the LP was achieved by incubatingthe protein in the presence of the LP at 37° C. for 30 minutes. Theprotein to lipid molar ratio was maintained for the first two trials ofpreparation (1:10,000). Additional, one trial using protein:lipid ratiosof 1:2000 were also investigated.

To estimate the amount of protein associated with LP, free protein wasseparated from LP-associated protein by floatation on a discontinuousdextran density gradient. Briefly, 0.5 ml of the LP-protein mixture wasmixed with 1 ml of 20% (w/v) dextran (in calcium free Tris buffer) in a5 ml polypropylene centrifuge tube and 3 ml of 10% (w/v) dextran and 0.5ml calcium free Tris buffer were overlaid on the LP-containing band. Thegradient was subjected to ultracentrifugation at 45,000 rpm for 30 minin a Beckman SW50.1 rotor. The LP and their associated protein floatedto the interface of the buffer/10% dextran bands, and the unassociatedprotein remained at the bottom. The concentration of the proteinassociated with LP was determined using spectroscopic assay. The percentassociation for protein:lipid of 1:10,000 was 63.9±9.6% (n=3) and thepercent association for protein:lipid of 1:2,000 was 40.1±1.1% (n=3).

Example 13

This example describes the application of this method to anotherprotein, lysozyme. The required amounts of DMPC, SPI and Chol(Dimyristoylphosphatidylcholine (DMPC):soy phosphatidylinositol(SPI):Cholesterol (Chol) (molar ratio 50:50:5) were dissolved inchloroform. A thin lipid film was formed on the walls of a glass tube,by removing the solvent in a Buchi-R200 rotoevaporator (FisherScientific). The lipidic particles (LP) were prepared by rehydration ofthe lipid film with 25 mM tris buffer (300 mM NaCl, pH=7.0; calciumfree) at 37° C. The LP were extruded twenty times through double stacked80 nm polycarbonate membranes using a high pressure extruder (LipexBiomembranes, Inc.) at a pressure of ˜200 psi. The size distribution ofthe particles was monitored using a Nicomp model CW380 size analyzer(Particle Sizing System).

The association of the protein with the LP was achieved by incubatinglysozyme in the presence of the LP at 37° C. for 30 minutes. The proteinto lipid molar ratio maintained at 1:2000 was investigated.

To estimate the amount of protein associated with LP, free protein wasseparated from LP-associated protein by floatation on a discontinuousdextran density gradient. Briefly, 0.5 ml of the LP-protein mixture wasmixed with 1 ml of 20% (w/v) dextran (in calcium free Tris buffer) in a5 ml polypropylene centrifuge tube and 3 ml of 10% (w/v) dextran and 0.5ml calcium free Tris buffer were overlaid on the LP-containing band. Thegradient was subjected to ultracentrifugation at 45,000 rpm for 30 minin a Beckman SW50.1 rotor. The LP and their associated protein floatedto the interface of the buffer/10% dextran bands, and the unassociatedprotein remained at the bottom. The concentration of the proteinassociated with LP was determined using spectroscopic assay.

Protein:Lipid Composition (molar ratio) % Association 1:2,000 DMPC (100)47.5 1:2,000 DMPC:SPI:Chol (50:50:5) 81.9

The particle thus prepared packaged the protein inside the particle andshields from the surrounding milieu. This is supported by acrylamidequenching data as shown in FIG. 6. This is likely to provide in vivostability as it may shield the protein from protease degradation.

Example 14

This example describes the association of another protein,erythropoietin (EPO) with the lipidic particles of the presentinvention. DMPC, SPI and Chol (Dimyristoylphosphatidylcholine (DMPC):soyphosphatidylinositol (SPI):Cholesterol (Chol) (molar ratio 50:50:5) weredissolved in chloroform. A thin lipid film was formed on the walls of aglass tube, by removing the solvent in a Buchi-R200 rotoevaporator(Fisher Scientific). The lipidic particles (LP) were prepared byrehydration of the lipid film with 25 mM tris buffer (300 mM NaCl,pH=7.0, calcium free) at 37° C. The LP were extruded twenty timesthrough double stacked 80 nm polycarbonate membranes using a highpressure extruder (Lipex Biomembranes, Inc.) at a pressure of ˜200 psi.The size distribution of the particles was monitored using a Nicompmodel CW380 size analyzer (Particle Sizing System).

The association of the protein erythropoietin with the LP was achievedby incubating EPO in the presence of the LP at 37° C. for 30 minutes.The protein to lipid molar ratio was maintained as 1:10,000 (3 trials)and 1:2000 (1 trial).

To estimate the amount of protein associated with LP, free protein wasseparated from LP-associated protein by floatation on a discontinuousdextran density gradient. Briefly, 0.5 ml of the LP-protein mixture wasmixed with 1 ml of 20% (w/v) dextran (in calcium free Tris buffer) in a5 ml polypropylene centrifuge tube and 3 ml of 10% (w/v) dextran and 0.5ml calcium free Tris buffer were overlaid on the LP-containing band. Thegradient was subjected to ultracentrifugation at 45,000 rpm for 30 minin a Beckman SW50.1 rotor. The LP and their associated protein floatedto the interface of the buffer/10% dextran bands, and the unassociatedprotein remained at the bottom. The concentration of the proteinassociated with LP was determined using spectroscopic assay.

The association efficiency as determined by fluorescence spectroscopywas 74.90%, 68.60% and 68.50% for a protein:lipid ratio of 1:10,000 and51% for a protein; lipid ratio of 1:2,000.

While the invention has been described through specific examples,routine modifications will be apparent to those skilled in the art andsuch modifications are intended to be within the scope of the invention.

1. Lipidic particles comprising phosphatidylcholine (PC),phosphatidylinositol (PI) and cholesterol, wherein the ratio of PC to PIis between 70:30 to 30 to 70 and cholesterol is present between 1 to 33%of PC and PI together, the particles have a size of between 40 to 140nm.
 2. The particles of claim 1, wherein the PC to PI ratio is between60:40 to 40:60.
 3. The particles of claim 1, wherein the PC to PI ratiois between 55:45 and 45:55.
 4. The particles of claim 3, wherein the PCto PI ratio is 50:50 and cholesterol is between 5-15% of PC and PItogether.
 5. The particles of claim 1, wherein each acyl chains of PCand PI independently has between 12 and 22 carbon atoms, and issaturated or unsaturated.
 6. The particles of claim 1, wherein theparticles are present in a lyophilized form.
 7. The particles of claim1, wherein the PI is soy PI and PC is egg PC.
 8. A compositioncomprising the lipidic particles of claim 1, and further comprising oneor more therapeutic agents associated therewith such that theimmunogenicity of the therapeutic agent is reduced, wherein thetherapeutic agent is a peptide, polypeptide or a protein.
 9. Thecomposition of claim 8, wherein the therapeutic agent is a proteininvolved in the blood coagulation cascade.
 10. The composition of claim9, wherein the therapeutic agent is selected from the group consistingof Factor VIII, Factor VII, Factor IX, Factor V, Willebrand Factor (vWF)and von Heldebrant Factor (vHF).
 11. The composition of claim 8 whereinthe therapeutic agent is selected from the group consisting of tissueplasminogen activator, insulin, growth hormone, erythropoietin alpha,VEG-F, thrombopoietin and lysozyme.
 12. The composition of claim 8,wherein at least 50% of the lipidic particles have a size of 140 nm orless.
 13. The composition of claim 12, wherein at least 60%, 70% 80% or90% of the lipidic particles have a size of 140 nm or less.
 14. Thecomposition of claim 12, wherein at least 50%, 60%, 70%, 80% or 90% ofthe lipidic particles have a size between 40 to 100 nm.
 15. Thecomposition of claim 12, wherein the size of the lipidic particles isbetween 80 and 100 nm.
 16. A method of reducing the immunogenicity of atherapeutic agent selected from the group consisting of a peptide,polypeptide or a protein comprising the steps of: a) preparing thelipidic particles of claim 1 by extruding multilamellar vesiclescomprising PC, PI and cholesterol through a sizing device to formlipidic particles of less than 140 nm; and b) mixing the therapeuticagent with the lipidic particles prepared in step a) wherein theimmunogenicity of the therapeutic agent is reduced.